The broad objective of this proposal is to characterize the interactions between the LexA repressor and the RecA nucleoprotein filament that leads to LexA autocleavage during the induction of the SOS response. This binding has been highly conserved among prokaryotes as evidenced by the fact that the RecA and LexA proteins from the distantly related E. coli and B. subtilis species apparently interact with each other equally well. Using the RecA and LexA proteins from both B. subtilis and E. coli, we propose to map the sites on both proteins that interact with one another. We have identified eleven amino acids in the LexA protein that significantly reduce RecA promoted LexA autodigestion, but have little or no effect on LexA catalytic activity. Using steady state kinetic analyses in vitro and kinetic studies in vivo we will identify LexA amino acids that are involved in binding the activated RecA filament. Once we have determined which amino acids in LexA are required for RecA binding, we will use site-specific crosslinking and mass spectrometry to identify the amino acid residues in RecA that are required for binding to specific amino acids in the LexA repressor. When the interaction sites are mapped on each of the proteins, we will align the two proteins in silico using the crystal structures of the E. coli RecA and LexA proteins;we will then test the computer model using further site-specific mutagenesis of the two proteins guided by the alignment. PUBLIC HEALTH RELEVANCE: Recent studies showing that the SOS response, and the corresponding increase in mutagenesis and release of mobile genetic elements, is activated by a variety of antibiotics in several different bacterial species suggests that the interaction between LexA and the RecA nucleoprotein filament may be the first step in generating resistance to certain antibiotics. Thus, understanding the precise nature of this interaction may facilitate the identification of potential therapeutic drugs that could be used to mitigate the evolution of antibiotic resistance.